Epigenomic response to albuterol treatment in asthma-relevant airway epithelial cells

Abstract

Background: Albuterol is the first-line asthma medication used in diverse populations. Although DNA methylation (DNAm) is an epigenetic mechanism involved in asthma and bronchodilator drug response (BDR), no study has assessed whether albuterol could induce changes in the airway epithelial methylome. We aimed to characterize albuterol-induced DNAm changes in airway epithelial cells, and assess potential functional consequences and the influence of genetic variation and asthma-related clinical variables.

Results: We followed a discovery and validation study design to characterize albuterol-induced DNAm changes in paired airway epithelial cultures stimulated in vitro with albuterol. In the discovery phase, an epigenome-wide association study using paired nasal epithelial cultures from Puerto Rican children (n = 97) identified 22 CpGs genome-wide associated with repeated-use albuterol treatment (p < 9 × 10^-8). Albuterol predominantly induced a hypomethylation effect on CpGs captured by the EPIC array across the genome (probability of hypomethylation: 76%, p value = 3.3 × 10^-5). DNAm changes on the CpGs cg23032799 (CREB3L1), cg00483640 (MYLK4-LINC01600), and cg05673431 (KSR1) were validated in nasal epithelia from 10 independent donors (false discovery rate [FDR] < 0.05). The effect on the CpG cg23032799 (CREB3L1) was cross-tissue validated in bronchial epithelial cells at nominal level (p = 0.030). DNAm changes in these three CpGs were shown to be influenced by three independent genetic variants (FDR < 0.05). In silico analyses showed these polymorphisms regulated gene expression of nearby genes in lungs and/or fibroblasts including KSR1 and LINC01600 (6.30 × 10^-14 ≤ p ≤ 6.60 × 10^-5). Additionally, hypomethylation at the CpGs cg10290200 (FLNC) and cg05673431 (KSR1) was associated with increased gene expression of the genes where they are located (FDR < 0.05). Furthermore, while the epigenetic effect of albuterol was independent of the asthma status, severity, and use of medication, BDR was nominally associated with the effect on the CpG cg23032799 (CREB3L1) (p = 0.004). Gene-set enrichment analyses revealed that epigenomic modifications of albuterol could participate in asthma-relevant processes (e.g., IL-2, TNF-α, and NF-κB signaling pathways). Finally, nine differentially methylated regions were associated with albuterol treatment, including CREB3L1, MYLK4, and KSR1 (adjusted p value < 0.05).

Conclusions: This study revealed evidence of epigenetic modifications induced by albuterol in the mucociliary airway epithelium. The epigenomic response induced by albuterol might have potential clinical implications by affecting biological pathways relevant to asthma.

Keywords: Airway cells; Albuterol; CREB3L1; DNA methylation; EWAS; Epigenetics; KSR1; MYLK4; Puerto Ricans; β2-agonist.

Fig. 1

Outline of the laboratory experiments design. A Air–liquid interface (ALI) mucociliary airway epithelia were generated from basal stem cells obtained from nasal brushing. Paired samples were stimulated with albuterol (Trx) and vehicle control (MeOH). B Experiment timeline. Basal airway epithelial cells were differentiated for 24 days (yellow), followed by 5 days of either control vehicle or albuterol stimulation (green). Cultures were harvested after 48 h of rest from the last stimulation (blue)

Fig. 2

Manhattan plot of the EWAS of DNAm changes induced by albuterol treatment in nasal epithelial cells. The blue and red lines represent the false discovery rate (FDR) < 5% and the genome-wide (p < 9 × 10^–8) significance thresholds, respectively. Gene annotation is represented for genome-wide significant CpGs, highlighting in boldface those CpGs that were validated

Fig. 3

Violin plots of independent meQTLs in nasal samples identified for the genome-wide associated CpGs that were validated: A cg23032799 (CREB3L1), B cg00483640 (MYLK4-LINC01600), and C cg05673431 (KSR1). The normalized ΔDNAm values (y-axis) are plotted against the genotypes for each independent SNP

Fig. 4

Bar plots of the significant results identified in the enrichment analyses using the Enrichr tool. The −log₁₀(q-value) for each term is represented in the x-axis. Only the top-three significant terms of the drug signature enrichment analyses are reported.